Abstract
Removal of pre-mRNA introns is an essential step in eukaryotic genome interpretation. The spliceosome, a ribonucleoprotein performs this critical function; however, precise roles for many of its proteins remain unknown. Genome-wide consequences triggered by the loss of a specific factor can elucidate its function in splicing and its impact on other cellular processes. We have employed splicing-sensitive DNA microarrays, with yeast open reading frames and intron sequences, to detect changes in splicing efficiency and global expression. Comparison of expression profiles, for intron-containing transcripts, among mutants of two second-step factors, Prp17 and Prp22, reveals their unique and shared effects on global splicing. This analysis enabled the identification of substrates dependent on Prp17. We find a significant Prp17 role in splicing of introns which are longer than 200nts and note its dispensability when introns have a < or =13-nucleotide spacing between their branch point nucleotide and 3 ' splice site. In vitro splicing of substrates with varying branch nucleotide to 3 ' splice site distances supports the differential Prp17 dependencies inferred from the in vivo analysis. Furthermore, we tested the predicted dispensability of Prp17 for splicing short introns in the evolutionarily distant yeast, Schizosaccharomyces pombe, where the genome contains predominantly short introns. SpPrp17 was non-essential at all growth temperatures implying that functional evolution of splicing factors is integrated with genome evolution. Together our studies point to a role for budding yeast Prp17 in splicing of subsets of introns and have predictive value for deciphering the functions of splicing factors in gene expression and regulation in other eukaryotes.
Highlights
Removal of pre-mRNA introns is an essential step in eukaryotic genome interpretation
DNA processing and repair defects are seen in alleles of CLF1/ SYF3 and PRP19 [13, 14]. Such overlapping effects could arise from the direct role of a factor in two or more cellular processes or could be a secondary consequence arising from the lack of a spliced mRNA for a gene product functioning in another pathway
Increased Precursor and Decreased Message Levels Serve as Global Indicators of Splicing Defects—We have examined the genome-wide effects of splicing factor mutations utilizing DNA microarrays containing all known and predicted S. cerevisiae ORFs and spots representing exclusively the predicted intron sequences
Summary
Growth Conditions, and Plasmids—Table I lists the strains and plasmids used here. They contained DNA spots for all the annotated yeast ORFs (as in the Sacchoromyces Genome Database) and, in addition, DNA spots for all the predicted introns in genes on the Watson strand (ϳ50% of all introns). Photostimulated luminescence counts for the pre-mRNA and mRNA were obtained in a phosphorimager These values, normalized to U5 small nuclear RNA levels, were log-transformed to the base 2 and plotted after zero transformation to the values at 23 °C (0 min time point). A second statistical analysis probed further into this bias by testing several nucleotide lengths, for these intron distance features, as discriminators of the affected or unaffected transcript groups. This was compared with the observed value of the number of affected introns, in a mutant, on either side of that specific cutoff
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